Cord blood transplantation (CBT) is an increasingly effective treatment for patients with hematological malignancies for whom suitable HLA-matched donors are not available. If T cells that mediate graft verus leukemia (GVL) could be distinguished from those that mediate graft versus host disease (GVHD), more effective immunotherapy strategies could be developed. Our long-term goal is to improve the outcome of CBT for patients with AML by engineering donor T cells to increase GVL without increasing GVHD. We hypothesize that donor T-cells targeting leukemia-associated antigens (LAA), such as the HLA-A2-restricted PR1 peptide on AML, preferentially mediate GVL activity over GVHD and that cord blood (CB) donor-derived PR1-specific cytotoxic T lymphocytes (PR1-CTL) can be elicited and expanded ex vivo for clinical use to selectively induce GVL in CBT recipients. Thus, we have cloned high- and low-affinity PR1-specific T cell receptor-alphaBeta (TCR) heterodimers from PR1 vaccine clinical responders, which can be transduced into polyclonal T-cells to redirect antigen specificity and mediate antileukemic effects. We have also produced a monoclonal antibody with high affinity for a specific conformational epitope of PR1/HLA-A2 (8F4) that mediates potent and specific cytotoxicity against acute myeloid leukemia (AML), and a single chain Fv of 8F4 fused with CD3zeta + CD28 as a chimeric antigen receptor (CAR) will be used to gene modify T-cells to study GVL effects. In addition, the number of precursor PR1-CTL is ~1000-fold higher in CB compared to adult peripheral blood and CB PRI-CTL can be activated and expanded more than 5-fold in vitro. On the strength of these advances, we propose to (1) identify an optimal method to elicit and expand potent CB-derived PR1-CTL ex vivo by comparing (a) cell expansion from single CB units, (b) cell purification from multiple donors, (c) PR1-TCR-alphaBeta gene modification, and (d) 8F4-CAR gene modification; (2) use a xenogeneic mouse model to validate the potency of PR1-CTL against human AML in vivo to study the persistence and possible tolerance induction of PR1-CTL by AML, and to determine the spatial and temporal GVL effects, persistence, and possible tolerance of CB-derived PR1-CTL using bioluminescence and PET/CT imaging. Finally, based on the method identified to optimally obtain PR1-CTL, we will (3) test the clinical feasibility and safety of CB-derived PR1-CTL as adoptive cell therapy for AML patients after CBT.